Assay of Pulmonary Microvascular Endothelial Angiotensin-Converting Enzyme in Vivo: Comparison of Three Probes

Abstract

We monitored the activity of pulmonary microvascular endothelial-bound angiotensin-converting enzyme (ACE) in vivo by means of multiple indicator-dilution-type techniques, utilizing three different probes: the hydrolysis of two substrates, [ 3H]benzoyl-Phe-Ala-Pro (BPAP) and [ 14C]benzoyl-Ala-Gly-Pro (BAGP), and the binding of the inhibitor [ 3H]RAC-X-65 (RAC), all measured during a single transpulmonary passage in anesthetized rabbits, placed on total heart bypass, so that both systemic and pulmonary circulations were fully supported by means of a two-channel extracorporeal pump. Experiments were performed at pulmonary blood flows ( Q b) of 250, 400, 560, and 800 ml/min in control or indomethacin-pretreated rabbits. ACE activity was also compared to that of pulmonary microvascular endothelial-bound 5′-nucleotidase, by measuring the dephosphorylation of its natural substrate 5′-[ 14C]AMP. We calculated substrate utilization, mean lung transit time (i), and volume of distribution (i.e., central blood volume) of all substrates, as well as inhibitor binding. We also calculated A max/ K m and B max products of enzyme mass and kinetic constants for substrates and inhibitor, respectively. As Q̇ b increased, A max/ K m values for all three substrates and B max increased linearly, indicating microvascular recruitment. In experiments in which either BPAP and 5′-AMP metabolism or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between Q̇ b-induced changes in A max/ K m values of BPAP vs 5′-AMP as well as in A max/ K m of BAGP vs B max of RAC. Similarly, increasing Q̇ b increased central blood volume and decreased t . Indomethacin had no effect on most of the hemodynamic or enzyme parameters measured. We conclude that in vivo assays of ACE proceed as predicted by Michaelis-Menten kinetics and offer insights into pulmonary endothelial pathophysiology.